This application addresses the broad Challenge Area (15) Translational Science, and specific Challenge Topic, 15-CA-102: Understanding mechanisms of hormone refractory cancers for therapeutic targeting. The development of hormone resistance in breast cancer is a significant clinical problem. The enhanced expression of the ErbB2/3 heterodimer leads to the ability of breast cancer cells to bypass normal endocrine responsiveness. However, the use of ErbB2 targeted- agents such as Trastuzumab for hormone resistant disease in the clinic has been disappointing. Thus, new strategies that reduce the activity of the ErbB2/3 heterodimer are urgently needed for treatment of hormone refractory breast cancer. EBP1, a protein isolated in our laboratory by its binding to ErbB3, is a negative regulator of ErbB2/3 signal transduction due to its ability to downregulate ErbB2 protein levels. Preclinical studies from our laboratory demonstrate that ecoptic expression of EBP1 increases sensitivity of breast cancer cells to the antiestrogen tamoxifen. Further, depletion of EBP1 protein by shRNA or inactivation of EBP1 by phosphorylation by PAK1, a kinase implicated in both breast cancer progression and tamoxifen resistance, results in development of the hormone refractory phenotyope. These data suggest that EBP1, by decreasing ErbB2 protein levels, can diminish ErbB2/3 receptor signaling that leads hormone resistance. We propose that EBP1 based therapies may be effective in reversing hormone resistance. Our ongoing in vitro and in vivo data indicate that an understanding of EBP1's mechanism of action will illuminate ErbB2 regulation and thus offer a new avenue for treatment of hormone refractory breast cancer. The aim of this proposal is to understand the mechanism by which EBP1 decreases ErbB2 protein levels and how deletion or inactivation of EBP1 contributes to hormone resistance. In addition, we hypothesize that PAK1 directed therapies may reverse hormone resistance in part via their ability to restore EBP1 function and reduce ErbB2 levels. We anticipate that these studies will lead to new therapies for treatment of ErbB2+, hormone resistant breast cancer. In Specific Aim 1, we will determine the mechanism of EBP1's attenuation of ErbB2 levels with the ultimate goal of developing EBP1 based therapies to downregulate ErbB2 expression. As several independent groups have demonstrated that EBP1 is both a DNA and RNA binding protein, we will examine effects on EBP1 on ErbB2 transcription and post-transcriptional events. We will therefore a) determine EBP1 induced changes in ErbB2 transcription using both promoter reporter and ChIP assays b) measure EBP1-induced changes in ErbB2 mRNA stability. We will extend studies indicating EBP1 binds ErbB2 mRNA and determine the region of EBP1 critical for RNA binding and the effects of phosphorylation of EBP1 on such binding and ErbB2 mRNA stability. The region of ErbB2 mRNA that binds EBP1 will also be determined and the function of this region in maintaining ErbB2 mRNA stability assessed. In Specific Aim 2, we will determine how to overcome PAK1 inactivation of EBP1 function for treatment of hormone refractory cancer. In Specific Aim 2a, we will demonstrate that PAK1 disables EBP1 and contributes to tamoxifen resistance. First, the ability of PAK1 to abrogate the EBP1 downregulation of ErbB2 mRNA and protein levels will be assessed. We will also determine if an EBP1 that cannot be phosphorylated by PAK1 can rescue PAK1 induced tamoxifen resistance. In Specific Aim 2b, we will test the ability of PAK1 inhibitors to ameliorate the hormone refractory phenotype via restoration of EBP1 activity. The effect of PAK1 inhibitors on EBP1 phosphorylation, tamoxifen sensitivity and ErbB2 levels in hormone resistant cell lines will be tested in vitro. In addition, the ability of combinations of PAK1 and ErbB2 inhibitors to reverse hormone resistance will be tested in vitro based on the hypothesis that a combined approach to target the ErbB2 receptor may be efficacious. Finally, we will confirm in vitro results by demonstrating the importance of EBP1 for tamoxifen sensitivity in animal models. These studies will then form the basis for in vivo trials of PAK1 inhibitors in tamoxifen resistant breast cancer cells. We suggest that EBP1 can form the basis of a new class of therapeutic agents for treatment of ErbB2+, hormone refractory breast cancer. Drugs can be based either on the structural components of EBP1 or on PAK1 inhibitors that will reactivate EBP1 function. EBP1 based therapies, in conjunction with currently available ErbB2 targeted drugs, may offer a new avenue for the development of treatments to reverse hormone resistance. PUBLIC HEALTH RELEVANCE: Despite numerous advances in therapies for breast cancer in recent years, recurrence and mortality remain high. Deregulation of the ErbB receptor family, a group of enzymes which stimulates cell growth, leads to resistance to chemotherapy and hormone treatment. We have cloned and characterized EBP1, an ErbB binding protein that decreases levels and activity of ErbB receptors, thus restoring sensitivity to hormone treatments. The current study is designed to determine how EBP1 regulates ErbB expression and hormone sensitivity. Our studies would establish EBP1 as a new therapeutic agent in breast cancer to restore hormone sensitivity in cells which overexpress ErbB receptors.